KR101123976B1 - Method for the quantitative assessment of global and specific DNA repair capacities of at least one biological medium, and the applications thereof - Google Patents

Abstract

Quantitatively evaluating the global and specific capacities for DNA repair in a biological medium, is new. Process for quantitative evaluation of the global and specific capacities for DNA repair in a biological medium comprises: (a) preparing a series of plasmids (P), each containing distinct DNA lesions, by independently treating them with a physical and/or chemical agent and recovery of the supercoiled fraction; (b) characterizing the lesions in each P; (c) depositing P, and at least one control supercoiled plasmid without lesions, on a single solid support, according to a pre-established scheme, to produce a functional support divided into x distinct zones (x = number of media to be tested simultaneously) where each zone contains the set of P; (d) incubating the support with different repair solutions, each containing the enzyme activities required for repair, ATP (adenosine triphosphate) and a system for regenerating it, a labeled nucleotide triphosphate and other components required for enzymatic activity, at 30 degrees Centigrade for 1-5, preferably 3, hours, with each repair solution being applied to a different zone A1-Ax; (e) washing the support; (f) (in)direct measurement of the signal produced by the label, incorporated into DNA during the repair reaction, in each zone; (g) recording and quantifying the signal from each plasmid in each zone; and (h) determining the ratio of signal from each plasmid relative to that from the control.

Description

Method for the quantitative assessment of global and specific DNA repair capacities of at least one biological medium, and the applications etc.

The present invention relates to a method for quantitative assessment of the overall and specific DNA repair capacity of a medium by evaluating the deletion / resynthesis capacity of a biological medium, and is also involved in its application.

DNA is constantly undergoing an endogenous or exogeneous attack resulting in the formation of base or sugar lesions.

The damage includes the following:

Damage to purine or pyrimidine bases: oxidative damage induced by cell metabolism and photosensitization; Damage through the production of chemical adducts resulting from the detrimental action of many genotoxic reagents such as polycyclic hydrocarbons; Injury through the formation of metheno-bases or etheno-bases;

Damage to the DNA double helix structure: cisplatin and insertion reagents, which form stable covalent bonds between bases carried by ultraviolet irradiation (cross-linking between pyrimidines) or by opposite strands (usually the formation of an intrastrand (between two adjacent bases of the same strand) or an interstrand caused by a bifunctional anticancer agent such as intercalating agents;

Spontaneous damage due to the fact that DNA is a partially unstable molecule: spontaneous deaminoation or depurination;

Damage via single-stranded or double-strand breakage: produced by agents such as ionizing radiation and through free radicals;

Sugar damage: destruction of deoxyribose resulting in breakage of phosphodiester bonds at the site of damage following strand breakage.

Diversity of induced damage is explained by analyzing stable photoproducts in which the following UVC irradiation was measured: pyrimidine (6-4) pyrimidone between two adjacent pyrimidines in contact with pyrimidine dimers due to the formation of cyclobutane rings Is formed. The relative proportion of pyrimidine dimer and (6-4) product is in the range of 10-4. In addition, their respective effectiveness in the lethal and mutagenic effects of ultraviolet irradiation is different: the dimers have a larger cytotoxic role than the (6-4) product, while the opposite for the mutagenic effect. . Similarly, ionizing radiation (e.g., γ-rays from cobalt 60) results in single-stranded or double-strand breakage (at a ratio of approximately 9: 1), many base addition products, base loss and high In DOS, crosslinking occurs between the DNA and the proximal protein (eg, chromosome) simultaneously. On average, one strand break is calculated per modified base. The predominant role of double-strand breakage in the cytotoxic effects of radiation involves the mutagenic effect due to base alteration.

These various types of damage can be produced on isolated DNA. For example, (6-4) photoproduct-type damage and cyclobutane-type pyrimidine dimers are induced by UVC irradiation (Hoeijmaker et al., Mutation Res ., 1990, 236, 223-238); Oxidative-type damage is induced by the Fenton's reaction in the presence of hydrogen peroxide and iron (Eliot et al., Free rad. Bio ; Med., 2000, 1438-1446). Another method of preparing modified DNA is to process plasmids by molecular biology techniques, insert oligonucleotides obtained by chemical synthesis, and include damage of interest. (Biade et al., J. Biol. Chem ., 1997, 273, 898-902).

All living organisms have a DNA repair system that seeks to maintain the integrity of their genes.

Of these repair systems, two have the ability to remove modified bases from DNA: they are base excision repair (BER) systems and nucleotide excision repair (NER) systems:

The BER system is more specifically provided for the repair of small damages in DNA such as oxidative damage, abasic sites, base fragmentations, base methylation, eteno-bases, and the like.

The NER system consists of acetylaminofluorine-DNA, cisplatin-DNA and psoraren-DNA adducts, dimers derived from UVB and UVC irradiation of DNA, covalent formation between DNA base and another molecule. Treats bulky injuries, leading to distortion of DNA double helix such as damage, etc. (Sancar et al. Annu. Rev. Genetics , 1995, 29, 69-105).

These various recovery systems have common characteristics, and in particular the following steps:

Recognition of damage by proteins in the repair system,

Damage and, optionally, cleavage of adjacent nucleotides,

Resynthesis of lost nucleotides by polymerase in the medium,

Repair is usually terminated by ligation of existing DNA strands with newborn strands.

In all such cases, the process involves incorporation, such as removal of modified nucleotides and replacement of one or more nucleotide triphosphates present in the repair medium in the DNA chain.

However, in particular, eukaryote repair systems are very complex and there are variants of this very simplified configuration (overall repair, repair involving DNA transcription, repair related to DNA replication, etc.). Notice the point. Some proteins are involved in multiple repair systems simultaneously, others are specific in a single system, some proteins can be induced by cytoplasmic or external factors, and others have ubiquitous constant expression.

The term "substrate" refers to any DNA that can undergo a repair reaction in the presence of DNA extracts and damage to the kidneys by the kidneys.

"Biological medium" or "cell extract" refers to being purified by an unpurified biological preparation that may contain one or more enzymatic activities involved in DNA repair.

The lesion can be associated with a particular protein that is largely responsible for its repair in DNA. Differences exist between species; Enzymes are less specific in prokaryotes such as Escherichia coli, while much more stringent lesion-specific repair enzyme association is observed in humans, especially in the BER system. For example, Lindahl and Wood explain that the BER system enzyme is the most important in humans and also important in related lesions (Science, 1999, 286, 1897-1905). For example, OGG1 protein, a glycosylase belonging to the human BER system, is involved in the repair of 8-oxo-2'-deoxyguanosine. In Escherichia coli, formamidopyrimidine-DNA N-glycosylase repairs this same lesion, but more commonly recovers oxidized purine bases (Seeberg et al., TIBS, 1995, 20, 391-397). Human protein ANPG is equivalent to bacterial protein AlkA. However, these enzymes do not have the same affinities for their substrates and have different ablation rate constants (Laval et al., Mut. Res., 1998, 402, 93-102). More than about 40 other lesions treated by the BER system can have negative biological consequences that cannot be ignored if they are not repaired. The enzymes responsible for their repair are considered to be potent anticancer agents. A clear knowledge of their substrate specificity can seem very important.

Recovery capability analysis (assays) of the cells have been developed and can be classified into two categories: in vivo (in which in vitro analysis requiring the use of activated cell extracts (in vitro assays), and carried out in living cells in vivo or semi in vivo system.

I. Methods Based on Determination / Resynthesis Activity Measurements

A. Most in vitro assays are based on the experiments described in Wood et al. (Cell, 1998, 53, 97-106 and Biochemistry, 1989, 26, 8287-8292), which assess the deletion / resynthesis stage of the repair process. Developed.

More specifically, this assay involves the use of plasmid DNA induced damage (pyrimidine dimers by UV irradiation, formation of crosslinks; single-strand removal or breakage through the action of DNase I); The DNA thus modified is incubated at 30 ° C. in the presence of a repair preparation comprising at least the cell extract to be evaluated, nucleotide triphosphate labeled with P at ^32- alpha and ATP. Enzymes contained in the extract incise the plasmid DNA to remove the lesion. DNA is newly synthesized by replacement of the removed nucleotides. Radionucleotides introduced into the medium are inserted into the DNA during the synthesis process. After separation by agarose gel electrophoresis of the recovered plasmid, the amount of radioactivity inserted, which is proportional to the recovery rate of the substrate, is measured. The method of preparing the cell extract and the reaction conditions affect the quality of the recovery step. In particular, the best repair yield appears to be obtained with whole cell extracts of the type used for ex vivo transcription, while extracts of the cytoplasm of the type used to enhance plasmid replication from the SV40 origin, and also other unpurified. Crude cell extracts exhibit nuclease activity that prevents correct interpretation of the repair phase.

Under conditions determined by Wood et al., The specificity of the reaction with respect to the irradiated DNA is greater at KCl concentrations on the order of 40-100 mM. Moreover, the irradiated DNA replication that occurs during repair maintains a constant ATP concentration, and more specifically in terms of the involvement of the cleavage step of repair, the presence of ATP and the ATP-producing system (phosphate creatine + creatine force). Highly dependent on pokinase). For example, the dependency does not occur in the case of strand breakage recovery. Control samples consisting of the same unmodified plasmid are used simultaneously in the reaction mixture.

Analysis by Wood et al. Requires the use of radiolabels, which adds a limitation that limits the implementation of these methods in routine analysis; Moreover, this analysis is not sufficient for simplicity and practicality in everyday use.

Wood et al. Have been proposed in assays to characterize extracts originating from cells established from patients with xeroderma pigmentosum (Satoh et al., Proc. Natl. Acad. Sci. USA, 1993 , 90, 6335-6339; Jones et al., Nucl.Acids Res., 1992, 20, 991-995; Robins et al., EMBO J., 1991, 10, 3913-3921). Pyrophilosis is a multigenic, multiallelic, autosomal recessive disease. Cells originating from patients with this disease are very sensitive to ultraviolet radiation and exhibit DNA repair defects. Eight genes are involved in various complementation groups of this disease: XPA to XPG and variant group XPV. Each group has different properties regarding DNA repair and in particular the various NER subtypes. Whether in the small damage category or in the broad category of damage, DNA lesions repair differently depending on the complementarity group.

In addition, other repair diseases (Cockayne syndrome, Ataxia Telangextasia) have specific repair characteristics and have been studied by Wood et al.

B. In various documents, the teams of B. Salles and P. Calsou (Biochimie 1995, 77, 796-802; Anal. Biochem. 1995, 232, 37-42) have identified plasmids attached to the wells of microplates. A method for detecting DNA lesions by carrying out a deletion / resynthesis reaction in a phase is described. The plasmid is taken up in the microplate wells and then modified with the chemical reagent posteriori . Cell extracts are added to the wells with digoxigenin-labeled nucleotide triphosphates. If damage cleavage occurs during the resynthesis step, the label is inserted into the DNA. The label is then revealed in each well by an antibody bound to alkaline phosphatase. Substrates that become luminescent after dephosphorylation with alkaline phosphatase are added to each well. The luminescence signal emitted from each well is measured. It is proportional to the insertion rate of the label.

This method is also described in international patent WO 96.28571, in which the inventors belonging to the team of B. Salles and P. Calsou describe a method for qualitatively and quantitatively detecting DNA lesions, wherein the damaged DNA may The composition comprising the cell extract to be attached to the support and tested and containing the label is contacted with the damaged DNA (prior to or following the step of attaching the solid to the support). They consider their method to be able to handle multiple samples simultaneously; Referring to the examples, repair in cell extracts comprises 150 μg protein, 50 mM KCl, 5 mM magnesium chloride, DTT, creatine phosphate, creatine phosphate kinase and various dNTPs, dNTPs to be labeled with deoxygenin The extract is carried out in 50 μl of reaction medium. The recovery is obtained after 3 hours of incubation at 30 ° C. and the wells are washed with a wash solution containing the phosphate buffer with salts, in which a nonionic surfactant (Tween 20) is added at a rate of 0.05 and 0.15% (preferably Composition: 10 mM phosphate buffer, 137 mM NaCl and 0.1% Tween 20). This assay is described as very sensitive in terms of the detection solution being performed on 40 ng of DNA instead of 200 or 300 ng of the assay solution.

Analysis by the team of B. Salles and P. Calsou suggests that the plasmid should be modified after attachment to a solid support. Among the damages generated by many chemical or physical reagents include chain breakage. This breakage is repaired very quickly and effectively by active cell extracts. Therefore, it is impossible to distinguish breakage recovery from recovery of other damages with this analysis. Breakage repair may even mask the repair of other damage and may interfere with signals resulting from the repair of damage to other DNA. Thus, it is a system that allows the detection of the overall effect without identification of the damages recognized by the repair system; In addition, the purpose of this method is not to detect and quantify the activity of proteins involved in DNA repair but to identify the presence of damage on the treated DNA.

II. Method based on evaluation of incision / excision steps

A variation of the method by Wood et al. Has been proposed, which makes it particularly possible to measure only damage incision activity.

A. Redaelli et al. (Terat. Carcinog. Mut., 1998, 18, 17-26) describe a method for direct culturing of plasmids with extracts without nucleotide triphosphate. Cleavage in the supercoiled plasmid causes a change in the rate of migration in the agarose gel during electrophoresis. Supercoiled plasmids move faster than incised plasmids due to their shape. Bands corresponding to various types of plasmids are quantified; The amount of the incised form correlates with the lesion incision activity of the plasmid containing the extract.

More specifically, this document cleaves 3 'or 5' positioned deoxyribose phosphodiester bonds of non-basic sites, thereby glycosyls specific for the modification to be repaired (alkylation, hydrolysis deaminoation, oxidation, mismatching). The cleavage activity of AP-endonuclease, which occurs on the basic site, obtained after the action of the raise is studied. In this document, the AP-endonuclease activity has been studied more clearly on crude extract of human leukocytes. First, the extract (80 μl) was incubated with intact plasmid (control), and second, incubated with depurinated plasmid. Thus, it was found that it is possible to quantitatively assess the action of AP-endonucleases as long as the incision activity depends on injury and is sensitive to EDTA.

The team of B. P. Calsou and B. Salles ( Biochem. Biophys. Res. Com ., 1994, 202, 788-795) proposes another approach to more clearly measure the incision activity of lesions of plasmids. After the first deletion step, they insert an inhibitor of aphidicolin specific to eukaryotic polymerase into the repair medium to prevent resynthesis by endogenous polymerase of normal DNA fragments. Polymerases of exogenous prokaryotic cells were mixed with the reaction medium in an amount equivalent to all test tubes. Thus, the differences in the results obtained reflect the damage cleavage step and do not reflect the resynthesis of the cleaved DNA fragments.

Another method based on the modification of the C. comet assay (gel electrophoresis in alkaline medium of single cell) also allows the measurement of incision activity. It was developed by Collins et al. (Mutagenesis, 2001, 16, 297-301). Oxidative damage is inserted into the gene DNA by photosensitization of HeLa cells in visible light. The cells are then bound to an agarose gel spread on the microscope slide, after which the cell membrane and protein are removed by controlled lysis. Nucleotides isolated from the gel are incubated in the active cell extract in the first injury incision step. The slide is then subjected to electrophoresis in an alkaline medium. The presence of cleavage generally leads to faster migration than nucleotides. The ball of DNA then intacts the appearance of the comet, the DNA containing the cleavage in the head, at the tail of the comet. The proportion of DNA in the tail of the comet, as determined by the specified software, is directly related to the incision activity contained in the extract used for the under consideration damage. This assay has been applied to measure cleavage activity of oxidative damage in extracts derived from human white blood cells. Compared to the method for measuring cleavage of plasmids, described by Redaelli et al. In 1998, Collins et al. Use a comet method to evaluate strand cleavage, which is first significantly more sensitive (every 10 ⁹ D). Detection of approximately 0.2 to 2), and secondly it is economical because the volume of the reaction mixture (DNA contained in the gel) is only 50 μl and a sufficient amount of material to be analyzed is obtained from 10 ml of blood (possibly carrying out several cultures). It is considered to have an advantage (savings by the materials used).

D. International patent WO 01/90408 describes a method for detecting and characterizing the activity of proteins involved in DNA repair.

More specifically, this method involves the attachment of one or more damaged DNA containing one or more known damages to a solid support; This damaged DNA then acts on a repair composition with or without one or more proteins involved in the repair of this damaged DNA and changes the signal emitted by a label attached or removed from the support during the previous step. By measuring this protein activity is determined for recovery.

Thus, this system, used with damaged DNA in the form of oligonucleotides of 15 to 100 bases or polynucleotides of 100 to 20,000 bases, is a different assay because cleavage of several substrates can be measured simultaneously. To get more overall information.

However, this method is involved in the demonstration of DNA lesion incision activity. Thus, characterizing the cleavage step of damage that can be inserted into the oligonucleotides of the synthesis is limited. Moreover, although it provides considerable information involved in cleavage activity, it is not appropriate for DNA deletion / resynthesis and does not describe an accurate quantitative assessment of enzymatic activity for DNA deletion / resynthesis.

In addition to the specific disadvantages for each of the techniques described above, these various methods also have the following disadvantages:

All assays described above require the use of a number of biological materials and in particular cell extracts larger than 10 μl: The reaction volume used is usually 50 μl containing 10-40 μl of extract in an amount of approximately 100 μg protein. to be. The extract takes a long time to prepare, and the amount of cells available is often small, which limits the number of assays that can be performed.

Whatever method used and performed in solution or support, all such systems described are involved in point-by-point recovery that is limited to a given substrate in aliquot fractions of extracts. Provide information; This is because, for example, as suggested by Wood et al., In assays that determine repair capacity, each assay is performed separately in vitro, ie the reaction occurs in a given plasmid and a given extract. The label insertion rate into the plasmid for each extract to be tested is compared with the insertion rate of the label obtained on the substrate prepared by the same method in the control extract. Reference control extracts are generally prepared from characterized cells transformed with EBV or SV40. The same is true for most other variations of the method described by Wood et al., Described above.

The experimenter limits the number of substrates used and the number of biological extracts tested, because much effort is required to perform the assay and requires the availability of large quantities of biological material.

Injuries inserted in the plasmid are neither measured nor quantified. The authors, using the assay developed by Wood et al., Only remove plasmids that have lost their supercoiled form to remove DNA containing chain breakage. The information obtained is very partial and insufficient to accurately define and characterize the recoverability of a given biological medium.

Consequently, the Applicant has evaluated to characterize and quantify the activity of the enzyme for the deletion / re-synthesis (excision / resynthesis) for DNA recovery from biological extracts in an efficient way the fast, accurate and compact without any particular use of the control group recovery solution It proposes a method which makes it possible to provide an object to overcome the disadvantages of the prior art.

An object of the present invention is a method for the quantitative assessment of the overall and specific DNA repair capacity of one or more biological media, characterized in that the method comprises the following steps:

(a) a set of plasmids comprising repairing distinct DNA lesions and individual supercoiled fractions of the plasmids by individual treatment of various plasmids with one or more physical and / or chemical agents (a range of plasmid) preparation: selection of supercoiled fractions blocks strand breakage and avoids the action of nucleases,

(b) distinguishing damages present on each plasmid of the plasmid set,

(c) According to a pre-established arrangement A, the various plasmids of the plasmid set and one or more supercoiled control plasmids without lesions are deposited on a single solid support to deposit another region A _1. To form a functionalized support divided by A _x , where x corresponds to an integer corresponding to the number of biological media tested at the same time, and each region A ₁ to A _x comprises said set of plasmids box); As a result, various samples of the plasmids of the set of modified plasmids are attached to defined and precisely designated sites on the support of the same solid. Attaching together one or more controls containing a supercoiled plasmid free of DNA lesions,

(d) incubating the functionalized support obtained in step (c) with various repair solutions; Prior to the incubation, each repair solution is attached at different predetermined regions A ₁ to A _x of the functionalized support, preferably for 1 to 5 hours, preferably for 3 hours, at 30 ° C. Incubated at a temperature, wherein each repair solution comprises one or more biological media including enzyme activity for repair, ATP, ATP-regenerating system, labeled nucleotide triphosphate and activity of repair enzyme present in the biological medium. Contains any other ingredients needed for.
(e) washing the functionalized support at least once,

delete

(f) in each different and predetermined region A ₁ to A _x , directly or indirectly measuring the signal generated by a label incorporated in DNA during the repair reaction in step (d),

(g) recording and quantifying the signal corresponding to each plasmid attachment in each region A ₁ to A _x ,

(h) determining the signal ratio of the lesion containing plasmid to the control plasmid attached together.

The method according to the invention has several advantages:

Due to the possibility of simultaneously evaluating the repair of various types of damage, the invention makes it possible to detect the overall effect during the identification of the various damages.

The present invention makes it possible to determine the cleavage and / or deletion / resynthesis capacity of biological extracts without resorting to comparison with the control biological medium. This is because the results obtained by performing the method with a single sample of biological extract are sufficient to give the extract a repair effect against accurate and quantified damage.

The present invention is particularly suitable for studying various biological media and is a good reflection of the in vivo environment.

The method according to the invention makes it possible to "map" a given biological medium in terms of its enzymatic activity for DNA repair. It is possible to identify biological extracts according to the map obtained.

The present invention makes it possible to determine repair proteins that are deficient or partially deficient in a given biological extract, and thus can serve as diagnostic tests.

Furthermore, the method according to the invention makes it possible to compare the performance levels of various biological extracts in terms of DNA lesion repair.

The present invention does not use any radioisotopes.

Since the present invention is miniaturized, it is possible to obtain a great deal of information using very small amounts of biological material.

The invention can be automated.

According to a preferred embodiment of the method, the plasmid prepared in step (a) is selected from those having a double-stranded supercoiled form (pBR322, M13, pUC, etc.).

The supercoiled form of the control plasmid is obtained by purification using the prior art, for example the Qiagen plasmid purification kit. It is also desirable to limit unwanted plasmids by performing other purification steps, eg cesium chloride centrifugation and / or sucrose gradient centrifugation.

According to another preferred embodiment of the method step (a), the various physical, biological or chemical reagents capable of inducing DNA lesions are preferably formed of a single lesion, a limited number of lesions or belonging to the same family. Selected from those that induce the formation of lesions.

As a group of lesions, mention may be made, for example, of photoproducts, chemical adducts, eteno-bases, non-base sites and DNA breakage induced by oxidative damage, ultraviolet B or C irradiation.

Physical and chemical reagents are selected, for example, mainly from those which function as follows.

By the photosentization mechanism II: the main target of singlet oxygen is guanine; In this case, many of the lesions formed are 8-oxoguanine (Ravanat et al., Chem. Res. Tox., 1995, 8, 379-388);

By photosensitization mechanism I or by mechanisms releasing OH ⁰ radicals; In this case, the DNA lesion obtained is an oxidative lesion; These lesions affect purine bases and pyrimidine bases in DNA in an equivalent manner. Among these lesions, 8-oxoguanine, glycols, thymine, papy-guanine, papy-adenine, hydroxymethyl-uracil, 5 5-hydroxymethylcytosine and formyluracil may be mentioned (cadet et al., Rev. Physiol. Biochem. Pharm., 1997, 31, 1, 87);

By the mechanism of triplet-triplet energy conversion; In this case, the main damage formed is cyclobutane pyrimidine dimer (Costalat et al., Photochem, Photobiol., 1990, 51, 255-262);

In the case of releasing energy absorbed directly by DNA base such as ultraviolet B or C irradiation. Bonds formed are cyclobutane pyrimidine dimers, (6-4) photoproducts and Dewar valence isomers (Douki et al., J. Biol. Chem., 2000, 275, 11678-11685);

For monolithic oxygen release. For example, such reagents belong to the group of endoperoxides. In this case, the lesion formed is 8-oxoguanine (Ravanat et al., J. Biol. Chem., 2001, 276, 40601-40604).

Chemical reagents are selected from those belonging to the group of so-called carcinogens that induce known base modifications. For example, acetaminofluorene (Hess et al., 1996, Nucleic Acid Res. 24, 824-828), cisplatin (Phsheva et al., 2002, Int. J. Biochem. Cell Biol., 34, 87- 92), benzopyrene (Laws et al., 2001, Mut. Res., 484, 3-18), psoralen (Zhang et al., Mol. Cell. Biol., 2002, 22 , 2388-2397), chloroactaldehyde (CAA-Wang et al., 2002, 13, 1149-1157), tamoxfen (Dasaradhi et al., 1997, Chem. Res. Tox., 10 , 189-196) and trans, trans-2,4-decadienal (DDE-Carvalho et al., 1998, Chem. Res. Tox., 11, 1042-1047) ) May be mentioned.

According to another preferred embodiment of step (a) of the method, various reagents are used on each plasmid of the plasmid set.

According to a preferred embodiment of step (b) of the method, the nature of the injury is i) obtaining a fraction of each plasmid with damage, (ii) said fraction with an enzyme that releases nucleosides from DNA. Digesting each of and then (iii) analyzing the result of degradation using a combination of independent techniques combined with quantitative analytical techniques.

According to a preferred arrangement of this embodiment, the degradation is carried out with the following enzymes: calf spleen phosphodiesterase, P1 nuclease, snake venom phosphodiesterase phosphodiesterase), and at least one of alkaline phosphotase (Douki et al., J. Biol. Chem., 2000, 275, 11678-11685).

According to another preferred combination of this embodiment, the results of the enzymatic digestion are described in the following technique: high performance liquid chromatography (HPLC) combined with tandem mass spectrometry (Douki et al., 2000, J. Biol. Chem., 275, 11678-11685; Sauvaigo et al., 2001, Photochem.Photobiol., 73, 230-237; Frelon et al., Chem. Res. Tox., 2000, 13, 1002-1010), gas chromatography HPLC coupled with chromatography (Wang et al., 2000, 13, 1149-1157; Pouget et al., 2000, Chem. Res. Tox., 13, 541-549) or electrochemical detection Analyzed by one of HPLC (Pouget et al., 2000, Chem. Res. Tox., 13, 541-549).

 According to another preferred embodiment of the method, before step (c), the supercoiled form of plasmid obtained in step (a) is purified by sucrose gradient centrifuge and / or cesium chloride gradient centrifuge.

According to another preferred embodiment of the method, prior to step (c) and also wherein each plasmid of the plasmid set preferably comprises a buffer at a pH of 6.5 to 8.0 optionally associated with salts and nonionic surfactants. Dilute to a concentration of 5-100 μg / ml in dilution buffer; Preferably, the buffer is 10 mM phosphate buffer or SSC buffer which may contain 0.05M to 0.5M NaCl.

Various plasmids are preferably attached using a robot intended for the production of microanalysis. That is, the volume of the deposits of the plasmid set is preferably 100 to 1000 pl (picoliter).

According to a preferred embodiment of step (c) of the method, the support is selected from the group consisting of organic or inorganic materials selected from glass, silicone and derivatives thereof, synthetic or non-synthetic polymers and optionally functionalized surfaces. , A support sensitized to increase affinity for DNA. Preferably, the support consists of a glass slide coated with poly-L-lysine to absorb DNA, or a glass slide functionalized with an epoxy group to form a covalent bond with the DNA.

If necessary, a treatment is performed to increase the adhesion of the DNA to its functionalized support. Such treatments should not create additional damage in the attached DNA.

A standard support according to the invention wherein each region comprises an A ₁ to A _x region comprising the entire set of plasmids, each of the regions comprising:

One or more control plasmid deposits and

Attachment and / or attachment of plasmids containing photoproducts

Attachment and / or attachment of plasmids containing oxidative damage

Attachment and / or attachment of plasmids containing etheno-bases

Attachment and / or attachment of plasmids containing DNA breaks

Attachment of plasmids containing carcinogen adducts.

According to step (d) of the method according to the invention,

Biological extracts are prepared according to the method of Manley et al. (Methods Enzymol., 1983 101, 568-582), or in a medium containing Biade et al. (J. Biol. Chem., 1998, 273, 898-902), or repair protein. Can be prepared according to any other method that can provide;

The label is selected from affinity molecules, fluorescent compounds, antibodies or biotin; Preferably, the label or reagent for visualizing the label is particularly selected from the group consisting of fluorescent compounds with direct fluorescent (Cy-3 or Cy-5) or indirect fluorescent (biotin or digogeninin);

The support is then incubated at a temperature that promotes the recovery reaction, preferably at 30 ° C., for a period between 1 and 5 hours, preferably 3 hours.

According to a preferred embodiment of step (e) of the process according to the invention, the support is washed at least once with brine containing a nonionic surfactant, in particular 10 mM phosphate buffer containing Tween 20, followed by water Rinse at least once.

According to step (f) of the method according to the invention, the signal is measured by a method suitable for labeling; For example, if the label is a fluorescent material, a direct measurement of the fluorescent signal emitted by the various precipitates on the support is performed.

According to a preferred embodiment of step (g) of the method according to the invention, the signal is quantified using a device capable of stimulating a label, preferably a fluorescent substance, and measuring the signal emitted by the stimulus .

The signal is measured by an instrument appropriate for the support and the label used. Scanners can be used for fluorescent image analysis, preferably with laser stimulation at a wavelength specific to the label used.

According to a preferred embodiment of step (h) of the method according to the invention, the proportion of the number of signals obtained from the plasmid containing the damage to the signal obtained with the control plasmid located on the same support is determined.

According to the present invention, a recovery profile of a given biological medium is obtained.

To determine the overall and specific repair capacity of the medium, to diagnose repair-related diseases, or to assess the impact of physical or chemical treatment (eg, genotoxic products) on the recovery capacity of a given medium. Profiles can be used.

As a result, it is an object of the present invention to use the method defined above:

-Determination of the recovery profile of the biological medium,

Diagnosing recovery-related diseases;

-Assessment of the impact of physical or chemical treatments on the recovery capacity of a given biological medium,

Screening of substances that can regulate the recovery system of the biological medium.

In addition to the above-described arrangements, the present invention may also include other combinations contemplated from the following techniques, where such techniques refer to embodiments of method implementation and accompanying drawings that are the object of the present invention:

1 shows an example of the support arrangement of solids; A plan of deposition in nine regions is observed;

2 shows the attachment surface of each region; And

FIG. 3 shows a recovery diagram—repair mapping associated with each cell line used to prepare the extract used for the recovery reaction.

However, it should be clearly understood that these embodiments are given merely by way of explanation of the purpose of the present invention and are not limited in any way.

Example 1 Preparation of a Set of Plasmids and Analysis of Lesions

Plasmid pBluescript II is generated by the transformation of XL1-Blue MRF supercompetent cells from stratazine, according to the protocol provided by Stratagene.

The plasmid is then purified using the Quiagen plasmid midi kit, according to the recommended protocol.

Further purification of the plasmid

The plasmid was added to 25 mM Tris HCl buffer, pH 7.5; 1M NaCl; Load in 10 ml of 5-20% sucrose gradient in 5 mM EDTA and centrifuge for 18 hours at 4 ° C. and 25000 rpm in a Beckman Ultracentrifuge using SW-41 rotor. 1 ml fractions are then carefully taken and analyzed on agarose gel. Only fractions containing at least 90% of the plasmid in coil form are retained. The plasmid is precipitated with ethanol and dissolved in PBS.

Plasmid modification

UVC irradiation-formation of cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts

Plasmids diluted to 20 μg / ml in PBS were irradiated using a bioblock germcidal lamp equipped with two 15-watt neons. The three plasmid plates were 0.06 each; Irradiation at 0.12 and 0.2 J / cm 2.

Treatment with chloroacetaldehyde (CCA-Signa) formation of malondialdehyde-dioxyguanine (MDA-dG)

The plasmid is prepared at 1 mg / ml in PBS and an equivalent volume of CAA (50% of water) is added. This solution is incubated overnight at 37 ° C. The plasmid is recovered by precipitation and purified on a sucrose gradient.

Treatment with trans, trans-2-4-decadienal (DDE-Sigma) formation of eteno-guanosine and eteno-adenosine

200 μl 0.2 M carbonate / bicarbonate buffer, pH 9.2 and equivalent volume of THF are added to 200 μl of plasmid prepared in water at 1 mg / ml. 4 μl of DDE and 12 μl of 30% H ₂ O ₂ are then added. The solution is incubated at 50 ° C. for 2 hours in the dark. DDE is removed by two extractions with dichloromethane. DNA is precipitated and then purified on sucrose gradient.

Treatment with endoperoxide DHPNO ₂ formation of 8-oxo-2′-dioxyguanosine (8-oxo-dG)

J. Biol. 20 μl of a solution of endoperoxide ( N, N′ -di (2,3-dihydroxypropyl) -1,4-naphthalenedipropanamide 1,4-endoperoxide) prepared according to the protocol described in Chem., 2000, 275, 40601-40604 Is incubated at 37 ° C. for 2 hours in 200 μl of plasmid diluted to 1 mg / ml in PBS. The plasmid is then precipitated and purified with sucrose gradient.

Analysis of lesions in plasmids

Fractions of plasmid DNA or calf thymus DNA treated under the same conditions were employed for the analysis of modified base composition.

The DNA is digested as described by Douki et al. (J. Biol. Chem., 275, 11678-11685), after which the analysis is performed by HPLC-tandem mass spectroscopy.

10 ^{4 The} following amount of damage was obtained per normal base:

process Etheno Da Etheno Dg MDA-
Dg 8-oxo-Dg Pyrimidine dimer (6-4)
Photo-Product Control DNA 0.11 0.00 0.00 0.61 Endoperoxide 0.01 0.00 0.02 6.15 DDE 1.42 9.04 0.24 2.89 UVC 0.06J / ㎠ 0.12 5.03 0.45 UVC 0.12 0.18 11.08 0.93 UVC 0.2J / ㎠ 0.21 18.14 1.57

 It should be noted that the treatment causes the formation of damage at very different rates.

The UVC irradiation causes the formation of cyclobutane pyrimidine dimers (CPDs) and a small range of (6-4) photoproducts very broadly,

The DDE causes the predominant formation of eteno-deoxyguanosine,

The endoperoxide causes a very predominant formation of 8-oxo-2'-dioxyguanosine.

It appears that such reagents make it possible to induce the predominant formation of specific damages that target the correct group of repair enzymes.

Example 2: Execution of the method according to the invention with a set of plasmids prepared in Example 1

Deposition of Plasmids on a Support

The plasmid is diluted to 20 μg / ml in PBS. On commercial poly-L-lysine-coated glass slides (VWR), 500 pl deposits are formed using a GESIM robot. The slide is stored at 4 ° C.

Each slide (support S) comprises nine identical areas A1 to A9 arranged in accordance with the arrangement A in FIG. 1.

In each region, a set of plasmids is attached, for example according to FIG. 2 which illustrates region A1.

Each region makes it possible to test different biological media.

Repair reaction

Solutions containing biological media and extracts to be tested are prepared; For 5 μl of solution, the composition is as follows:

0.5 μl extract

1 μl of 5 × recovery buffer

0.2 μl of CY5-dUTP (Amersham Pharmacia Biotech) (0.1 nmol / μl)

0.2 μl 2M KCl

0.1μl of ATP (Roche-100mM)

The volume is supplemented with 5 μl with water.

Composition of 5x Recovery Buffer:

pH 7.8, 200 mM Hepes / KOH); 35 mM MgCl ₂ ; 2.5 mM DTT; 2 μm dATP, 2 μm dGTP, 2 μm dCTP; 50 mM phosphocreatin; 250 μg / ml creatine phosphokinase; 0.5 mg / ml BSA; 17% glycerol.

Cell lines used

This example is performed with three different extracts from different cell lines:

? Cell line 1 : These are HeLa cells. The extract is a commercial nuclear extract and the manufacturer is 4C Biotech (Belgium). They were prepared by the method of Dignam et al. (Nucl. Ac. Res., 1983, 11, 1475-1489). Their protein content is 24 mg / ml.

? Cell line 2 : This is an AS203 cell line established from complementary group D, a patient suffering from xeroderma pigmentosum (complementation gruop D). Extracts were prepared according to the protocol of Manley et al. Analysis of proteins using the micro BCA kit allows for the evaluation of proteins in amounts of 44 mg / ml.

? Cell line 3 : These are XP12RO cells. This cell line was established from complementary group A, a patient suffering from xeroderma pigmentosum (complementation group A). Extracts were prepared according to the protocol of Manley et al. (Methods Enzymol., 1983, 101, 568-582). The extract obtained contains 36 mg / ml of protein (micro BCA assay kit, interchim).

3 μl of each recovery solution is attached onto all precipitates of the slide single zone. The slides are incubated at 30 ° C. for 3 hours under moist conditions. The slides are washed three times for 10 minutes in PBS buffer containing 0.1% Tween 20. Then wash it with water for 15 minutes. After drying, read the fluorescence.

Analysis of Recovery Signal

After the recovery reaction, the fluorescence of the various precipitates in each region is analyzed by Axon scanner and GenePix Pro analysis software at 635 nm. The mean of three identical points is then determined. The figures are obtained as a result of each type of deformation. Charts are drawn for each cell line. This diagram corresponds to the mapping of the repair system related to the damage present on the support or chip and is specific for the extract used. The results obtained are given in FIG. 3. The concentration of fluorescence is given in arbitrary units (AU).

Each plot was observed to be unique and specific to the cell line used to prepare the extracts used. Therefore, the plot can be used to accurately characterize the overall repair activity of a given cell extract and reveal the function of the targeted system.

It was observed that the HeLa cell line repairs DDE-induced damage (tapped etheno-dG) twice as effectively as repairing damage induced by UV irradiation (tapped CPD and (6-4)). It was observed that oxidative damage (tapped 8-oxo-dG) recovered much weaker.

For the AS203 cell line, although low, the highest degree of recovery was observed in UV-induced damage.

For the XP12RO cell line, it was observed that DDE-induced damage (tapped etheno-dG), which gives a signal three times higher than that of UVC irradiation, is repaired more effectively. It is known that XPA cell lines do not repair CPDs; Thus, the signal obtained with UVC-irradiated DNA can be attributed to the recovery of the (6-4) photoproduct.

An unexpected advantage of the present invention is that even if the amount of protein is different, the ratio obtained from the signal of the DNAs, including damage to the signal of the control DNA, can be used to compare the recoverability of various extracts.

Claims (23)

A method of quantitative assessment of the overall and specific DNA repair capacity of one or more biological media, comprising the following steps: (a) by treating the various plasmids individually with one or more physical reagents, one or more chemical reagents, or mixtures thereof and restoring the supercoiled fraction of each plasmid, each plasmid contains distinct DNA lesions. Preparing a set of plasmids, (b) characterizing the lesions present in each plasmid of the plasmid set, (c) According to the pre-established arrangement A, the various plasmids of the plasmid set and one or more supercoiled control plasmids without lesions are attached on a single solid support to other regions A ₁ to A _x Forming a functionalized support divided by x, wherein x corresponds to an integer corresponding to the number of biological media tested at the same time, and each region A ₁ to A _x comprises said set of plasmids, (d) incubating the functionalized support obtained in step (c) with a variety of recovery solutions for 1 to 5 hours at a temperature of 30 ° C., wherein each recovery solution has at least one biological medium capable of enzymatic activity for recovery. , ATP, ATP-regenerating system, labeled nucleotide triphosphate and other components necessary for the activity of repair enzymes present in the biological medium, wherein prior to the incubation, each repair solution is used to Applied to different predetermined areas A ₁ to A _x , (e) washing the functionalized support at least once, (f) in each of said other predetermined regions A ₁ to A _x , directly or indirectly measuring a signal generated by a label incorporated in DNA during the repair reaction in step (d), (g) recording and quantifying the signal corresponding to each plasmid attachment in each region A ₁ to A _x , and (h) determining the signal ratio of the plasmid comprising the lesion to the control plasmid attached together. The method of claim 1 wherein the plasmid according to step (a) is selected from those having a double-stranded supercoiled form. The method of claim 1, wherein the various physical or chemical reagents capable of inducing a lesion of DNA in step (a) of the method include: formation of a single lesion, formation of a limited number of lesions, or a variety belonging to the same family. And from those inducing the formation of lesions. The method of claim 1, wherein in step (a) of the method various reagents are used for each plasmid of the plasmid set. The method of claim 1 wherein characterizing the lesions in step (b) of the method comprises (i) obtaining a fraction of each plasmid with lesions, and (ii) the fractions with an enzyme that releases nucleosides from DNA. Digesting each of the following and then (iii) analyzing the digestion results using a combination of separate techniques combined with quantitative analysis techniques. The method of claim 5, wherein the digestion is performed by the following enzymes: calf spleen phosphodiesterase, P1 nuclease (P1 nuclease), snake venom phosphodiesterase, and alkaline Characterized in that it is carried out using one or more of alkaline phosphotases. The method of claim 5, wherein the results of the enzyme digestion are described in the following techniques: high performance liquid chromatography (HPLC) combined with tandem mass spectrometry, HPLC coupled with gas chromatography, or electrochemical detection Characterized in that it is analyzed by one of HPLC combined). The method of claim 1, wherein before step (c), the supercoiled form of plasmid obtained in step (a) is purified by a sucrose gradient centrifuge, cesium chloride gradient centrifuge, or a combination thereof. How to feature. The method of claim 1, wherein before step (c) and also each plasmid of the plasmid set is diluted with a salt and a nonionic surfactant to a concentration of 5-100 μg / ml. The method of claim 1, wherein in step (c) of the method, the deposits volume of the plasmid set is between 100 and 1000 pl (picoliter). The method of claim 1, wherein in step (c) of the method, the support is sensitized to increase affinity for DNA, comprising organic or inorganic materials selected from glass, silicon and derivatives thereof, and synthetic or nonsynthetic polymers. And a method selected from the group consisting of: The method of claim 11, wherein the support consists of a glass slide coated with poly-L-lysine that absorbs DNA or a glass slide functionalized with an epoxy group to form a covalent bond with the DNA. The method of claim 11, wherein the support comprises other regions A ₁ to A _x , wherein each region is: One or more control plasmid deposits, and Attachment of plasmids containing photoproducts, attachment of plasmids including oxidative damage, attachment of plasmids containing etheno-bases, attachment of plasmids including DNA breakage And at least one plasmid attachment selected from the group consisting of attachment of the plasmid comprising a carcinogen adduct: Method comprising a. The method of claim 1, wherein in step (e) the support is washed at least once with a saline solution containing a nonionic surfactant. The method of claim 14, wherein the support is washed in 10 mM phosphate buffer containing Tween 20 and then rinsed at least once with water. The method of claim 1, wherein in step (g) the signal is quantified using a device capable of exciting the label and measuring the signal emitted upon excitation. 2. The method of claim 1, wherein in step (h) of the method the numerical ratio of the signal obtained from the plasmid comprising the lesion to the signal obtained with the control plasmid placed on the same support is determined. 18. Use of the method according to any one of claims 1 to 17 to establish a repair profile of a given biological medium. 18. Use of the method according to any one of claims 1 to 17 to diagnose a repair-related disease. 18. Use of the method according to any one of claims 1 to 17 to assess the effect of physical or chemical treatment on the recoverability of a given medium. 18. Use of the method according to any one of claims 1 to 17 for screening substances capable of adjusting the recovery system of the biological medium. The method of claim 9, wherein each of the plasmids is diluted in a buffer having a pH of 6.5 to 8.0. The method of claim 11, wherein the surface of the selected support is functionalized.

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